Pharmacokinetics-Driven Optimization of 4(3 H)-Pyrimidinones as Phosphodiesterase Type 5 Inhibitors Leading to TPN171, a Clinical Candidate for the Treatment of Pulmonary Arterial Hypertension

Article abstract of DOI:10.1021/acs.jmedchem.9b00123

Phosphodiesterase type 5 (PDE5) inhibitors are first-line therapy for pulmonary arterial hypertension (PAH) and erectile dysfunction. As a continuing work to improve the terminal half-lives and oral bioavailabilities of our previously reported 4(3H)-pyrimidones, a pharmacokinetics-driven optimization focusing on the terminal substituent is described. Two major congeneric series of 4(3H)-pyrimidones, the aminosulfonylphenylpyrimidones and acylaminophenylpyrimidones, were designed, synthesized, and pharmacologically assessed in vitro and in vivo. Among them, compound 15 (TPN171) with subnanomolar potency for PDE5 and good selectivity over PDE6 was finally recognized as a potential drug candidate, and its pharmacokinetic profiles in rats and dogs are significantly improved compared to the starting compound (3). Moreover, TPN171 was proven to exert a longer lasting effect than sildenafil in animal models, providing a foundation for a once-daily oral administration for its clinical use. TPN171 is currently being investigated in a phase II clinical trial for the treatment of PAH.

Full text of DOI:10.1021/acs.jmedchem.9b00123

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Article
Pharmacokinetic-Driven Optimization of 4(3H)-Pyrimidinones as
Phosphodiesterase Type 5 Inhibitors Leading to TPN171, a Clinical
Candidate for the Treatment of Pulmonary Arterial Hypertension
Zhen Wang, Xiangrui Jiang, Xianglei Zhang, Guanghui Tian, Rulei Yang, Jianzhong Wu, Xiaoli Zou, Zheng
Liu, Xiaojun Yang, Chunhui Wu, Jing Shi, Jianfeng Li, Jin Suo, Yu Wang, Rongxia Zhang, Zhijian Xu,
Xudong Gong, Yang He, Weiliang Zhu, Haji Akber Aisa, Hualiang Jiang, Yechun Xu, and Jingshan Shen
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.9b00123 • Publication Date (Web): 25 Apr 2019
Downloaded from http://pubs.acs.org on April 25, 2019
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of their duties.

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Zhu, Weiliang; Shanghai Institute of Materia Medica Chinese Academy of
Sciences, Drug Discovery and Design Centre
Aisa, Haji Akber; Xinjiang Technical Institute of Physics and Chemistry,
Chinese Academy of Sciences, Xinjiang Key Laboratory of Plant
Resources and Natural Products Chemistry
Jiang, Hualiang; Shanghai Institute of Materia Medica Chinese Academy
of Sciences, Drug Discovery and Design Center; Shanghai Institute of
Materia Medica, Drug Discovery and Design Center
Xu, Yechun; Shanghai Institute of Materia Medica Chinese Academy of
Sciences, Drug Discovery and Design Center
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Shen, Jingshan; Shanghai Institute of Materia Medica Chinese Academy
of Sciences,
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Pharmacokinetic-Driven Optimization of 4(3H)-Pyrimidinones as
Phosphodiesterase Type 5 Inhibitors Leading to TPN171, a Clinical
Candidate for the Treatment of Pulmonary Arterial Hypertension
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†
,#
†,#
†,‡,#
§
§
Zhen Wang, Xiangrui Jiang, Xianglei Zhang, Guanghui Tian, Rulei Yang,
§
§




Jianzhong Wu, Xiaoli Zou , Zheng Liu, Xiaojun Yang, Chunhui Wu, Jing Shi,
†
†
†
†
†
†,
Jianfeng Li, Jin Suo, Yu Wang, Rongxia Zhang, Zhijian Xu, Xudong Gong,
†
†,‡

†,‡,*
Yechun Xu,†,‡,*and
Yang He, Weiliang Zhu, Haji Akber Aisa, Hualiang Jiang,
Jingshan Shen†,‡,*
†CAS Key Laboratory of Receptor Research, Drug Discovery and Design Center,
Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai
2
01203, China
‡School of Pharmacy, University of Chinese Academy of Sciences, Beijing 100049
§Vigonvita Life Science Co., Ltd., Suzhou 215123, China
Topharman Shanghai Co., Ltd., Shanghai 201203, China
Key Laboratory of Plant Resources and Chemistry in Arid Regions, Xinjiang
Technical Institute of Physics and Chemistry, Chinese Academy of Sciences, Urumqi
8
30011, China
ABSTRACT
Phosphodiesterase type 5 (PDE5) inhibitors are first-line therapy for pulmonary
arterial hypertension (PAH) and erectile dysfunction. As a continuing work to
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improve the terminal half-lives and oral bioavailabilities of our previously reported
4
(3H)-pyrimidones, a pharmacokinetic-driven optimization focusing on the terminal
substituent is described. Two major congeneric series of 4(3H)-pyrimidones, the
aminosulfonylphenyl pyrimidones and acylaminophenylpyrimidones, were designed,
synthesized and pharmacologically assessed in vitro and in vivo. Among them,
compound 15 (TPN171) with subnanomolar potency for PDE5 and good selectivity
over PDE6 was finally recognized as a potential drug candidate and its
pharmacokinetic profiles in rats and dogs are significantly improved compared to the
starting compound (3). Moreover, TPN171 was proved to exert a longer lasting effect
than sildenafil in animal models, providing a foundation for a once-daily oral
administration for its clinical use. TPN171 is currently being investigated in a phase II
clinical trial for the treatment of PAH.
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INTRODUCTION
Pulmonary arterial hypertension (PAH) is a progressive disorder characterized by
abnormally high blood pressure in the pulmonary artery that carries blood from the
heart to the lungs, ultimately leading to right heart failure and death.1-4 It is a rare
disease with a high mortality rate. Although survival of PAH patients is still
unsatisfactory and annual mortality from this devastating disease remains high (~10%
per year), the therapy for PAH has evolved progressively in the past decade.5, 6
Several effective drugs, including cyclic nucleotide phosphodiesterase-5 (PDE5)
inhibitors，a soluble guanylate cyclase stimulator, prostacyclin receptor agonists, and
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endothelin receptor antagonists, have been approved.7-11 For instance, sildenafil and
tadalafil, two inhibitors of PDE5 which is responsible for specifically cleaving cyclic
guanosine monophosphate (cGMP), have been approved by FDA for the treatment of
PAH.12-14 The pulmonary vasculature contains substantial amounts of PDE5, and
regulation of NO/cGMP signalling cascades by inhibition of this enzyme results in
decrease of the pulmonary pressure due to vasodilation of the pulmonary artery.15
PDE5 inhibitors also exert anti-proliferative effects on pulmonary artery smooth
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muscle cells. These features make an advantage of PDE5 inhibitors for the treatment
of PAH.17-19 In addition, the PDE5 inhibitor administration is not only taken as a
monotherapy but also is used in combination with other drugs such as the endothelin
receptor antagonist to improve long-term outcomes in PAH.20
We previously described the discovery of a series of 4(3H)-pyrimidinones as
novel, potent and selective inhibitors of PDE5 for a potential use in PAH as well as
male erectile dysfunction (ED).21-23 The eleven sub-families of human PDEs
(PDE1-11) possess a conserved C-terminal catalytic domain for hydrolysis of the
ubiquitous second messenger, cyclic adenosine monophosphate (cAMP) and cGMP.24
High selectivity is thus crucial to a PDE5 inhibitor as each PDE isozyme acts as a key
element in regulation of some physiological processes, and inhibition of other PDE
isozymes rather than PDE5 may lead to off-target-based adverse effects.25 Both
sildenafil and tadalafil served as effective therapeutics for PAH as monotherapy or a
key component of combination therapy, however, their clinical usefulness has been
limited due to the excessive side effects mostly derived from the unsatisfactory
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selectivity across multiple PDEs.26-29 In addition, the approved dose of sildenafil for
3
0
the treatment of PAH is 20 mg t.i.d because of its short half-life. Considering that
PAH is a chronic disease, it is of paramount importance to acquire a convenient and
safe oral once-daily therapy. Accordingly, on basis of a lead compound (3) from our
previously reported 4(3H)-pyrimidinones, we continued our project aiming to find a
novel PDE5 inhibitor with more potential therapeutic benefits in PAH treatment by
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improving the properties of pharmacokinetics as well as selectivity of the
compound.21
RESULTS AND DISCCUSSION
Design of Aminosulfonylphenyl Pyrimidones. Among our previously reported
inhibitors,
5,6-diethyl-2-[2-n-propoxy-5-(4-methyl-1-piperazinylsulfonyl)phenyl]pyrimid-4(3H)
-one) stands out as a highly potent and selective PDE5 inhibitor (IC =1.6 nM) and
compound
3
(
5
0
exhibits a good efficacy in the animal model. However, the pharmacokinetic study
revealed that the compound had an insufficiently long terminal half-life (t =1.29 h)
1
/2
and a poor oral bioavailability (F =10%) in rats, preventing it from being a candidate
for clinical studies.21 Our major efforts are thereby devoted to improve the
pharmacokinetic properties of this congeneric series. Earlier SAR studies have
already demonstrated that the 5,6-diethyl-pyrimidin-4(3H)-one of 3 exhibits the
strongest inhibition potency among the pyrimidin-4(3H)-one analogues and the
n-propoxyl substituent at the 2’-position of the phenyl ring contributes the favorable
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hydrophobic interactions with the enzyme. Moreover, the determined crystal structure
of the catalytic domain of PDE5 in complex with 3 (PDB code: 4G2W) indicates that
it fits well into the ligand binding pocket except for the methylpiperazine group,
which extended out into a solvent-exposed surface region and had rare interactions
with the protein surroundings (Figure 1A-C). It is in line with a sparse
electron-density associated with the methylpiperazine ring. From these observations,
we presumed that further modifications for improvement of the pharmacokinetic
properties should be performed on the methylpiperazine moiety to minimize the effect
on potency and selectivity of the compound.
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To test our conjecture, we first replaced the methylpiperazine ring of compound 3
with three different substituents, resulting compounds (4-6) (Scheme 1 & Table 1).
Opening of the methylpiperazine ring and attachment of a basic morpholine to keep
the three-bond linker between two nitrogen atoms resulted in compound 4. In
addition, an acidic carboxyl was introduced on the alpha and beta position of N atom
of sulfonamide to make compounds 5 and 6, respectively. An in vitro enzyme activity
assay showed that three compounds all retained the potency (IC of 1.01, 2.02 and
5
0
1
.69 nM for 4, 5 and 6, respectively) as high as that of 3, while their inhibition
selectivity over PDE6 varied (Table 1). Among them, compound 6 exhibits an
outstanding selectivity over PDE6 (86-fold), which is more than double of 3 (39-fold).
The in vitro human liver microsome (HLM) metabolic stability test has been
extensively used in drug discovery projects to evaluate the metabolic stability of a
compound in development. Such a test was thus performed on compound 6. To our
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surprise, no metabolism was found with 6 after 30-minute incubation of it with HLM
(HLM%=100%) while only 29% of 3 was remained at the same condition (Table 1),
suggesting that compound 6 is extremely stable in HLM. Higher stability as well as
better selectivity of 6 over compound 3 prompts us to carry out an investigation on the
in vivo efficacy of compound 6 in male spontaneously hypertensive rats (SHR) model.
Unfortunately, it does not show an expected efficacy in vivo (Figure 2A), possibly due
to the strong polarity of the carboxylic group resulting in poor membrane permeability
and oral absorption.
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Figure 1. Crystal structures of the catalytic domain of PDE5 in complex with
compounds 3 (green, PDB code: 4G2W) and 15 (magenta, PDB code: 6IWI). (A) The
Fo-Fc difference electron-density maps contoured at 3.0 σ for two compounds bound
in PDE5. (B) Molecular surface of the ligand binding pockets of PDE5 with two
compounds. (C, D) LIGPLOT representation of detailed interactions formed between
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the protein and compounds 3 (C) or 15 (D).31
Scheme 1. Synthesis of compounds 4-6.a
O
O
O
Et
Et
Pr-n
Et
Et
Pr-n
Et
Et
Pr-n
NH
O
NH
O
NH
O
a
b
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N
N
N
SO Cl
4-6
O S
2
1
2
2
R1
a
Reagents and conditions: (a) chlorosulfonic acid, 5 °C; (b) amines, Et N, dichloromethane.
3
Table 1. Chemical Structure, PDE5/PDE6 Inhibitory Activity and Metabolic
Stability in Human Liver Microsome (HLM%) of Aminosulfonylphenyl
Pyrimdinones (3-6)
O
Et
Et
Pr-n
NH
O
N
O S
2
R1
Compds
3
R1
IC50(PDE5) (nM)
IC50(PDE6)/IC50(PDE5)
HLM (%)
N
1.60
39
29%
N
CH3
O
N
4
5
6
1.01
2.02
1.69
10
20
86
/
/
N
OH
O
OH
N
O
100%
N
OH
H
Scheme 2. Synthesis of compounds 7-21.a
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O
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N
Et
Et
Pr-n
Et
Et
Pr-n
Et
Et
Pr-n
Et
Et
Pr-n
NH
O
NH
O
NH
O
NH
O
a
b
d
N
.
NO2
NH2 HCl
HN
R2
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10-14, 17-21
c
9
, base form of 8
comp. R2
comp. R2
O
O
N
O
O
Et
Et
Pr-n
Et
Et
Pr-n
10
17
NH2
NH
O
NH
O
O
O
N
e
N
1
1
2
18
N
Ac
O
O
HN
HN
Cl
R2
NH2
1
19
1
4
O
15, 16
N
O
O
O
N
O
O
comp. R2
comp. R2
H
N
N
20
21
13
O
N
O
O
N
N
S
15
16
14
Cl
O2
a
Reagents and conditions: (a) conc. H SO , conc. HNO , 5 °C, 90%; (b) H , 10%Pd/C, HCl gas,
2
4
3
2
methanol, r.t., 97%; (c) 0.4 N NaOH, 86%; (d) for 10-13, acids, Et N, EDCI, HOBT,
3
dichloromethane, r.t.; for 14, 18, 21, acyl chloride or methylsulfonyl chloride, Et N,
3
dichloromethane, 0 °C; for 17, KCNO, HOAc, reflux, 91%; for 19, 20, ethyl oxalyl monochloride,
Et N, dichloromethane, 0 °C, then amines, methanol; (e) amines, Et N, DMF, 60 °C.
3
3
Design of Aminophenylpyrimidones. Substitution of the methylpiperazine ring
confirmed that this moiety can be replaced by other groups without sacrificing the
potency. In addition, according to the crystal structure of PDE5 in complex with 3, the
sulfone group has no interaction with the protein and its orientation prevents the
methylpiperazine ring from contacting the protein surface, leading us to think about a
further replacement of the sulfone group. Considering a smaller size for less steric
obstruction and synthetic ease for further modification, an amine group was
introduced to replace the sulfone (Scheme 2 & Table 2). Compound 9, with an
unsubstituted amino group on the benzene ring, had single digit nanomolar potency
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against PDE5 (IC = 5.33 nM) and demonstrated a moderate selectivity over PDE6
5
0
(10-fold). As the amino group on a benzene ring often leads to adverse effect on
pharmacokinetic property and safety of the compound, a carbonyl connected with
different substituents were subsequently designed to yield amides 10-13, 15, and 16.
Except compound 10, a second nitrogen with a form of a tertiary amine or an amide
was introduced and the length of the linker between two nitrogen atoms varied in the
rest five compounds. Again, these compounds retain the high potency against PDE5,
in particular, most compounds exhibiting a subnanomolar potency (IC s: 0.54, 0.79,
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
5
0
0.92, and 0.62 nM for 11, 12, 13, and 15, respectively). The selectivity of these
compounds against PDE5 over PDE6 showed greater variability, ranging from 7 to
32-fold, and compound 15 demonstrated the best one with a 32-fold selectivity over
PDE6. The in vitro HLM metabolic stability test showed that 15 with a HLM% of
9
1% was much more stable than compound 3 after 30-minute incubation with HLM.
Therefore, compound 15 was selected for further investigation.
In addition, substitution at the NH group of 9 using other functional groups to
2
form urea (17-18), oxalic amide (19-20) and sulfamide (21) were carried out (Table
2
). As expected, these compounds have nanomolar or subnanomolar inhibitory
activities against PDE5 and moderate selectivity over PDE6. Among them, compound
21 shows a 28-fold selectivity over PDE6 but is unfortunately not so stable in HLM
(HLM% = 27%).
Table 2. Chemical Structure, PDE5/PDE6 Inhibitory Activity and HLM% of
Aminophenylpyrimdinones (9-21)
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9
O
Pr-n
NH
O
N
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
HN
R2
IC50(PDE6)/IC50(PDE5)
Compds
9
R2
IC50(PDE5) (nM)
HLM (%)
H
5.33
10
/
O
10
1.21
0.54
11
/
/
O
1
1
10
N
O
O
1
1
2
3
0.79
0.92
24
7
/
/
N
O
H
N
O
O
N
1
1
5
6
0.62
1.46
1.61
32
9
91%
N
O
/
/
N
O
17
17
NH2
O
1
1
8
9
1.86
0.83
11
22
/
/
N
O
NH2
O
O
N
N
2
2
0
1
1.55
1.23
17
28
/
O
S
27%
O
2
Structural and Thermodynamic Characterization of 15 Binding to PDE5. As
mentioned above, the introduction of the amino group instead of the sulfone aims to
1
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7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
reduce the steric obstruction and to establish more interactions of the substituent with
the protein surface. To test the idea, we determined the crystal structure together with
the thermodynamic profiles of the catalytic domain of PDE5 binding to compounds 3
and 15 (Tables 3-4 and Figure 1, PDB code: 6IWI). As expected, the carbonyl oxygen
of 15 adopted an orientation opposite to that of the sulfone oxygen of 3 so that the
following N,N-dimethylpiperazine moiety could lie down the protein surface and form
hydrophobic interactions with residues G819 and A823 (Figure 1). This provides an
explanation for the higher in vitro potency of 15 over 3 as the terminal
methylpiperazine ring of 3 has no interaction with the protein (Figure 1). Consistent
with the structure information, the binding enthalpy of 15 with PDE5 measured by
isothermal titration calorimetry (ITC) experiments is more significant compared to 3,
whereas the interactions established between the N,N-dimethylpiperazine group and
the surrounding residues lead to more entropic losses associated with 15 than with 3
(Table 4). Consequently, the total binding free energy of 15 with PDE5 is slightly
stronger compared to 3. Altogether, the structural and thermodynamic data of two
compounds is not only in line with their IC s but also gains deep insight into the
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
5
0
essential factor responsible for the different potency, rationalizing our strategy for use
of the amino group instead of the sulfone.
Table 3. Crystallography Data Collection and Refinement Statistics
Data Collection
PDB ID
PDE5/15
6IWI
Space group
Cell constants: a (Å)
b (Å)
P3121
74.75
74.75
1
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9
c (Å)
132.02
0.979
Wavelength (Å)
Resolution range (Å)
36.39 - 2.15 (2.25-2.15) a
Redundancy
18.9 (17.5)
I/σ (I)b
6.9 (3.2)
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Completeness (%)
Refinement
94.23 (57.88)
Rwork/Rfreec
0.185/0.227
22358
Reflections (unique)
RMS Values
Bond length (Å)
Bond angle (°)
Protein atoms
Ligands
0.002
0.435
2313
2
Water molecules
Clashscore
146
1.32
0.86
MolProbity Score
Ramachandran plot
Favored (%)
99.65
0.35
0.00
Allowed (%)
Outliers (%)
aStatistics for highest resolution bin of reflections in parentheses.
bIntensity signal-to-noise ratio.
cRfree is calculated against a 5% random sampling of the reflections that were removed before
structure refinement
Table 4. Thermodynamic Properties of Compounds 3 and 15 Binding to PDE5
Compds
3
K (M)
d
ΔG (kJ/mol) ΔH (kJ/mol)
-TΔS (kJ/mol)
30.49±3.45
IC (nM)
50
(1.31 ± 0.05) x 10-7 -39.86±0.09 -70.35 ± 3.43
(0.82 ± 0.03) x 10-7 -41.06±0.26 -90.07 ± 1.35
1.6
1
5
49.01±1.16
0.62
Inhibitory Selectivity of 15 Over 11 PDEs. We measured the selectivity profile
of compound 15 against 11 human recombinant PDEs (Table 5) by comparing its in
vitro potency for PDE5 to its inhibition of other PDEs. Sildenafil and tadalafil were
used as reference inhibitors and their selectivity indexes across 11 PDEs were also
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9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
evaluated in parallel. The IC values of 15, sildenafil and tadalafil toward PDE5 are
5
0
0
.62, 4.31, and 2.35 nM, respectively. Compound 15 showed an excellent selectivity
over PDE2, 3, 4, 7, 8, 9, and 10 (>16,129-fold). Remarkably, a selectivity of 15
toward PDE11 was found to be 1610-fold, much more selective than tadalafil (9-fold).
In addition, the selectivity of 15 over PDE1 as well as PDE6 was higher than
sildenafil (5871-fold vs. 190-fold to PDE1 and 32-fold vs. 8-fold to PDE6). Overall,
the potency and the selectivity of 15 over 11 PDEs are superior to those of sildenafil
and tadalafil, implying less side effects from the treatment with 15 can be anticipated.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Table 5. In Vitro Activities and Selectivities of 15, Sildenafil and Tadalfil to 11
PDE Enzymes (mean±SD, n=3)
1
5
Sildenafil
Tadalafil
PDEs
IC (nM)
Selectivity*
5871
IC (nM)
Selectivity* IC (nM)
Selectivity*
>4255
>4255
>4255
>4255
-
5
0
50
50
PDE1
PDE2
PDE3
PDE4
PDE5
PDE6
PDE7
PDE8
PDE9
PDE10
PDE11
3640±215
>10000
>10000
>10000
0.62±0.13
19.8±7.7
>10000
>10000
>10000
>10000
998±192
819±89
>10000
>10000
>10000
4.31±0.46
36.4.2±1.8
>10000
>10000
>10000
>10000
4930±1140
190
>2320
>2320
>2320
-
>10000
>10000
>10000
>10000
2.35±0.28
402±56
>10000
>10000
>10000
>10000
22.1±5.9
>16129
>16129
>16129
-
32
8
171
>16129
>16129
>16129
>16129
1610
>2320
>2320
>2320
>2320
1144
>4255
>4255
>4255
>4255
9
*
: Selectivity, IC50(PDEs)/IC50(PDE5)
Pharmacokinetic Profiles of 15. Pharmacokinetic profiles of compound 15 in rats
and dogs were comprehensively evaluated and summarized in Table 6. After oral
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administration of a 1 mg/kg dose of 15 to Sprague-Dawley rats (three males and three
females), it revealed a Tmax of 2.8 h, a Cmax of 43.9 ng/mL, a AUC of 268 ng/h/mL, a
0-t
AUC0-∞ of 284 ng/h/mL, a MRT of 5.56 h, a t1/2 of 2.82 h, and an oral bioavailability
of 66.4%. The same experiment with intravenous administration of 15 suggested that
it has a moderate plasma clearance (CL = 2.26 L/h/kg) and high volume of
distribution (Vss = 4.97 L/kg). Moreover, the pharmacokinetic profile of 15 with a
dose of 0.5 mg/kg in beagle dogs is more desirable than that in rats. After intravenous
administration to dogs (three males and three females), a much lower plasma
clearance (CL = 0.648 L/h/kg), and a similar volume of distribution (Vss = 3.83 L/kg)
were observed compared with the higher dose administration to rats, leading to a
longer terminal half-life (t1/2 = 4.89 h) in dogs than in rats. Additionally, 15
maintained the good oral bioavailability of 67% after oral administration to dogs. The
promising pharmacokinetic properties of 15 in rats as well as in dogs warrant further
evaluation of its therapeutic potential in PAH as well as other PDE5-associated
diseases.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Some pharmacokinetic data of sildenafil and tadalafil reported previously were
also listed in Table 6 for comparison. The terminal half-life of 15 in rats is longer than
sildenafil and is comparable to tadalafil. The oral bioavailability of 15 in rats is much
better than sildenafil and is similar to tadalafil. In addition, in healthy volunteers, the
terminal half-life of sildenafil and tadalafil after oral administration is 3.7 and 17.5 h,
respectively. Taking the preclinical and clinical pharmacokinetic data of sildenafil and
tadalafil together with the preclinical pharmacokinetic profile of 15, we speculated
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9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
that the human terminal half-life of 15 would be more than 10 hours. As anticipated,
in our phase I first-in-man clinical trials (Registration No.: CTR20171238,
http://www.chinadrugtrials.org.cn ), it did show a terminal half-life of 10.9 h after oral
administration of 5 mg of 15. Accordingly, the excellent pharmacokinetic data of
compound 15 lay a good foundation for a once-daily oral dosing profile of 15 for the
treatment of PAH.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Table 6. Pharmacokinetic Profiles of 15, sildenafil and tadalafil
Dose
T
max
C
max
AUC0-t
AUC0-C
MRT
t
1/2
CL
Vss
F
Compounds Species Route
(mg/kg)
(h)
(ng/mL) (ng/h/mL) (ng/h/mL)
(h)
(h)
(L/h/kg)
(L/kg) (%)
IV
Rats
1.0
/
/
403±47
412±51 2.22±0.23 2.4±0.37
284± 82 5.56±0.51 2.82±0.61
2.26±0.28 4.97±0.18
/
PO
1.0
0.5
0.5
4.0
1.0
/
2.8±0.4 43.9±11.6 268±83
/
/
66.4
1
5 a
IV
PO
IV
/
/
752±272 773±289 6.07±0.84 4.5±0.51 0.648±0.175 3.83±0.77
Dogs
1.9±0.7 66.5±14.1 495±148 514±164 7.21±1.05 4.89±1.09
/
1.83
/
/
1.6
/
67.0
34
/
1.6
/
/
76
/
/
/
/
/
/
/
/
/
/
/
/
/
1.1
0.65
2.4
/
Sildenafil b Rats
Tadalafil c Rats
PO
IV
0.31
/
5.2
/
PO
/
/
/
63
aPharmacokinetic profiles of 15 were determined as described in the EXPERIMENTAL
SECTION; data are expressed as mean±SD, n=6.
bPharmacokinetic profiles of sildenafil are taken from literature, and data are the arithmetic
average of the males and females.30
cPharmacokinetic profiles of tadalafil are taken from literature.13
In Vivo Efficacy of 15 in Animal Models
The in vivo efficacy of compound 15 was first investigated in an acute model of
1
4
hypertension in spontaneously hypertensive rats (SHR). Figure 2B shows that in the
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first two hours 15 and sildenafil exerted an equivalent effect on drop of blood pressure
of SHR, such a drop effect was further reinforced for 15 but was attenuated for
sildenafil in the next couple of hours, and the apparent antihypertensive effect of 15
maintained over 24 hours while sildenafil hardly had any efficacy after 6 hours.
Hence, treatment with 15 rapidly and remarkably decreases the blood pressure of
SHR and such an antihypertensive effect is kept for long time, gaining a much more
robust efficacy than sildenafil in SHR.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Subsequently, effects of compound 15 against PAH were evaluated in a
3
2
monocrotaline (MCT)-induced PAH rat model. Sildenafil and bosentan, targeting
the nitric oxide and endothelin pathways, respectively, are the first-line oral drugs for
the treatment of PAH. They were used in our studies as the positive control. After 21
consecutive days of injection of MCT, a significant increase of the mean pulmonary
artery pressure (mPAP) was observed in the model group compared with the control
group (p<0.01), demonstrating that the application of MCT successfully induced the
symptom of PAH. Treatment with compound 15, sildenafil and bosentan all
apparently decreased the mPAP of the rats (Figure 3). Administration of 15 showed
the efficacy comparable to that of sildenafil or bosentan, however, its dose (1 mg/kg)
was much lower than that of sildenafil (25 mg/kg) or bosentan (100 mg/kg), which is
principally owing to its high in vitro PDE5 activity. Taken together, these data
demonstrated the robust efficacy of compound 15 in two animal models and thus
promoted a further development of this compound into a clinically therapeutic
application.
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8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 2. Effect of compound 6 (A) and 15 (B) on mean arterial blood pressure in
SHR model (Mean ±SD, n=6). Sildenafil was used as a reference. Normal saline was
taken as vehicle.
6
5
0
0
#
#
*
***
***
40
3
2
0
0
10
0
C
M
B
S
1
1
5
o
o
o
il
n
d
s
d
(
1
tr
e
e
e
o
l
n
n
m
/k
l
ta
a
fi
g
n
l
/
(
(
2
k
g
0
5
)
0
m
m
g
g
/
k
g
g
)
)
Figure 3. Effects of 15 on mean pulmonary arterial pressure (mPAP) in MCT rats
model. Bosentan and sildenafil were used as references. The data are expressed as the
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mean ± SD (n = 20/group). ##: P<0.01 compared with the control group; *: P<0.05,
*
**: P<0.001 compared with the model group.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Chemistry
The synthesis of compounds 4, 5 and 6 was pursued with a similar protocol as
2
1
previously reported (Scheme 1). The key intermediate 2 was freshly prepared by the
treatment of 1 with chlorosulphonic acid at 0 °C.33 The sulfonylation reactions of
amines by 2 were accomplished with triethylamine as base in dichloromethane. The
selective nitration at the C5 position of benzene ring of compound 1 in the mixture of
concentrated sulfuric acid and concentrated nitric acid at 5 °C gave 7 with satisfying
yield and purity. The reduction of 7 in the methanol under hydrogen atmosphere gave
crude 9. The solution of crude 9 in methanol was treated by dried hydrogen chloride
gas to form 8 (salt form of 9), and the purification of 8 was achieved by the
crystallization in methanol. Purified 8 was alkalized in water to furnish 9 with high
purity (Scheme 2). Because 8 has better stability than 9 at the same storage condition,
it was used as the key intermediate for synthesis of compounds 10-21. Compounds
1
0-13 were prepared by condensation of 8 with propanoic acid, N-acetyl-L-proline, 1-
methyl-4-piperidinecarboxylic acid or 4-(acetylamino)-butanoic acid, respectively, in
the presence of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide and
-hydroxybenzotriazole. Compound 8 is acylated by chloroacetyl chloride to give 14,
1
which is the intermediate to produce compounds 15 and 16 by alkylation of the
N-methylpiperazine and piperidine, respectively. Compound 17 was obtained by a
reaction of 8 with potassium cyanate in the presence of acetic acid. Compound 8 was
1
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treated by pyrrolidine-1-carbonyl chloride and dichloromethane using triethylamine as
the base to furnish 18. 19 and 20 were furnished by acylation of 8 by ethyl oxalyl
monochloride, followed by ammonolysis reaction with ammonia or
N-methylpiperazine, respectively. Sulfonylation of 8 by methanesulfonyl chloride in
dichloromethane gave 21 (Scheme 2).
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CONCLUSIONS
Given the effectiveness of PDE5 as a key target for PAH, ED and new indications
discovered, both in academia and pharmaceutical industry there is continuing interest
in the discovery of novel PDE5 inhibitors with less side effects. Here, we present the
development of compound 3, a potent and selective PDE5 inhibitor reported
previously, into a clinical candidate (compound 15, also named TPN171) with a major
effort devoted to optimizing the pharmacokinetic profile of the compound. As design
of new compounds with desirable pharmacokinetic properties has proved challenging
and the crystal structure suggests that the methylpiperazine substituted sulfone of 3
hardly interacts with the enzyme, small modifications on this moiety were performed.
Two major congeneric series of 3, three aminosulfonylphenyl pyrimidones and
thirteen acylaminophenyl pyrimidones, were designed, synthesized and assessed in
terms of their PDE5 and PDE6 activities. All compounds retained the high potency
against PDE5 as their IC values range from single-digit nanomolar to subnanomolar.
5
0
Compounds only with selectivity over PDE6 which is comparable to or better than 3
were filtered by an in vitro HLM metabolic stability test, and the metabolically stable
compounds were further investigated in animals to evaluate the in vivo
1
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pharmacokinetics and efficacies for PAH. Through these efforts we were able to
identify TPN171 (compound 15), which possesses multiple drug-like properties such
as high potency, outstanding selectivity, well-characterized mechanism of action,
excellent pharmacokinetic profile, and easy preparation of the active pharmaceutical
ingredient (API), warranting further evaluation of its therapeutic potential in clinical
trials. A phase II clinical trial is now under way to test its safety and efficacy in PAH
patients.
1
1
1
1
1
1
1
1
1
1
2
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0
EXPERIMENTAL SECTION
All commercially available materials and solvents were used without any further
1
purification. TLC analyses were performed on Merck silica gel 60 F254 plates. H
1
3
NMR spectra and C NMR spectra were recorded at room temperature on a Bruker
AMX-400 using TMS as an internal standard. Mass spectra were recorded on a
Finnigan MAT-95/711 spectrometer. The purity of test compounds were determined
by HPLC (Agilent ChemStation, Aigent ZORBAX RX-C8 4.6*250mm, 5um, 30 °C,
flow rate = 1.0 mL/min) with aqueous CH3CN (25%−72%, 0.1% FA) for 30 min. The
purity of all synthesized compounds is not less than 95%.
General Procedure for the Synthesis of 4-6. Compound 1 (1 mmol) was slowly
added into chlorosulfonic acid (5 mL) at 5 °C, after stirred at room temperature for 2
h, the reaction mixture was added dropwise into brash ice to generate a faint yellow
precipitate. The solid was collected by filtration, washed with ice water and dried
under reduced pressure to give crude 2. The crude 2 was dissolved in dichloromethane
2
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(50 mL), and added dropwise into dichloromethane (30 mL) containing amine (1.1
mmol) and triethylamine (1 mL) at 5 °C, followed by stirring for 30 min, the organic
phase was washed with water (3×20 mL) and saturated brine (20 mL), and evaporated
off solvent to dryness to give the residue. Melting points were taken in open capillary
on a RY-1G apparatus and were uncorrected.
0
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0
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5,6-Diethyl-2-(2-n-propoxy-5-(N-(2-morpholinylethyl)-N-(2-hydroxyethyl)
aminosulfonyl)phenyl)-4(3H)-pyrimidone
(4).
The
amine
was
2
-(2-morpholinoethylamino)-ethanol, and the residue was recrystallized from ethyl
acetate/hexane to give the title compound to give off-white solid (291 mg, yield 56%
1
over two steps). mp 76-77 °C. H NMR (CDCl 500 MHz): δ 10.97 (1H, br), 8.88
3
,
(1H, s), 7.90 (1H, d, J = 8.5 Hz), 7.15 (1H, d, J = 9.0 Hz), 6.25 (1H, br), 4.25 (2H, t, J
=
6.0 Hz), 3.83 (2H, s), 3.76 (4H, s), 3.28 (2H, s), 3.22 (2H, s), 2.59-2.74 (10H, m),
13
2
.03 (2H, m), 1.28 (3H, t, J = 7.0 Hz), 1.16 (6H, m); C NMR (CDCl , 125 MHz): δ
3
164.45, 162.08, 160.13, 149.82, 131.87, 131.11, 130.59, 125.20, 120.55, 113.15,
7
1.91, 66.38, 62.85, 57.81, 53.41, 53.38, 47.30, 27.52, 22.26, 18.73, 13.31, 13.05,
+
10.57; LRMS m/z 523.4 [M+H] ; HRMS (ESI) m/z calculated for C H N O S
2
5
39
4
6
+
[
M+H] 523.2585, found 523.2594; HPLC purity 97.76%, t = 21.99 min.
r
N-(3-(5,6-Diethyl-4(3H)-pyrimidone-2-yl)-4-n-propxyphenylsulfonyl-L-prolin
e (5). The amine was L-proline, and the crude product was recrystallized from ethyl
acetate/hexane to give the title compound to give off-white solid (282 mg, yield 61%
1
over two steps). mp 172-174 °C. H NMR (CD OD 500 MHz): δ 8.32 (1H, d, J = 2.0
3
,
Hz), 8.05 (1H, dd, J = 8.5, 2.0 Hz), 7.36 (1H, d, J = 9.0 Hz), 4.22 (2H, t, J = 6.5 Hz),
2
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4.09 (1H, m), 3.55 (1H, m), 3.34 (1H, m), 2.73 (2H, q, J = 7.5 Hz), 2.62 (2H, q, J =
7
1
.5 Hz), 1.91 (5H, m), 1.65 (1H, m), 1.31 (3H, t, J = 7.5 Hz), 1.17 (3H, t, J = 7.5 Hz),
13
.08 (3H, t, J = 7.0 Hz); C NMR (CD OD 125 MHz): δ 164.24, 164.17, 164.08,
3
,
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
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3
3
3
3
3
3
3
3
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4
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5
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5
5
5
6
0
1
2
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5
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0
1
2
3
4
5
6
7
8
9
0
1
2
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6
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8
9
0
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160.21, 152.32, 132.09, 130.16, 129.94, 124.02, 121.57, 113.04, 71.03, 63.25, 48.87,
3
1.24, 26.69, 24.39, 22.01, 21.95, 18.11, 12.35, 9.49; LRMS m/z 464.3 [M + H]+;
+
HRMS (ESI) m/z calculated for C H N O S [M+H] 464.1850, found 464.1863;
2
2
30
3
6
HPLC purity 96.38%, t = 16.98 min.
r
2
-(N-(3-(5,6-Diethyl-4(3H)-pyrimidone-2-yl)-4-n-propoxyphenyl)sulfonyl)ami
nopropanoic acid (6). The amine was 3-amino-propanoic acid, and the crude product
was recrystallized from, which was recrystallized from ethyl acetate/hexane to give
the title compound as off-white solid (262 mg, yield 60% over two steps). mp
1
1
86-187 °C. H NMR (CD OD 500 MHz): δ 8.24 (1H, d, J = 2.5 Hz), 7.88 (1H, dd, J
3
,
=
8.5, 2.0 Hz), 7.24 (1H, d, J = 9.0 Hz), 4.12 (2H, t, J = 6.5 Hz), 3.02 (2H, t, J = 7.0
Hz), 2.61 (2H, q, J = 7.5 Hz), 2.50 (2H, q, J = 7.5 Hz), 2.37 (2H, t, J = 7.0 Hz), 1.80
1
3
(
2H, m), 1.19 (3H, t, J = 7.5 Hz), 1.05 (3H, t, J = 7.5 Hz), 0.96 (3H, t, J = 6.0 Hz); C
NMR (CD OD 125 MHz): δ 173.54, 164.37, 163.69, 159.99, 152.12, 132.58, 131.51,
3
,
1
9
29.56, 124.04, 121.53, 112.95, 71.06, 38.56, 34.05, 26.79, 21.94, 18.10, 12.31, 12.27,
+
.45; LRMS m/z 438.3 [M+H] ; HRMS (ESI) m/z calculated for C H N O S
2
0
28
3
6
+
[M+H] 438.1693, found 438.1704; HPLC purity 96.29%, t = 15.19 min.
r
2-(5-Nitro-2-n-propoxyphenyl)-5,6-diethyl-4(3H)-pyrimidone
(7).
The
compound 1 (40 mmol) was dissolved in concentrated sulfuric acid (100 mL) under
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ice-bath, and slowly added with concentrated nitric acid (100 mL). After stirred at
room temperature for 3h, the reaction mixture was slowly added dropwise into ice
water to generate a light yellow precipitate. After filtered, the solid was washed with
0
1
2
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4
5
6
7
8
9
0
1
2
3
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water (3×200 mL) and dried at 60 °C to give the title compound 7 as yellow solid
1
(
11.92 g, yield: 90%). mp 138-139 °C. H NMR (CDCl 500 MHz): δ 11.32 (1H, br),
3
,
9.20 (1H, d, J = 3.0 Hz), 8.34 (1H, dd, J = 9.0, 3.0 Hz), 7.16 (1H, d, J = 9.0 Hz), 4.28
(1H, t, J = 6.5 Hz), 2.71 (2H, q, J = 7.5 Hz), 2.58 (2H, q, J = 7.5 Hz), 2.02 (2H, m),
13
1
.31 (3H, t, J = 7.5 Hz), 1.14 (6H, m); C NMR (CDCl , 125 MHz): δ 164.68, 162.37,
3
161.43, 149.74, 141.84, 127.85, 127.26, 125.18, 120.85, 112.86, 72.16, 27.61, 22.22,
1
8.71, 13.27, 13.14, 10.48; LRMS m/z 332.3 [M+H]+.
2-(5-Amino-2-n-propoxyphenyl)-5,6-diethyl-4(3H)-pyrimidone
hydrochloride
(8)
and
2
-(5-Amino-2-n-propoxyphenyl)-5,6-diethyl-4(3H)-pyrimidone (9). The compound
7 (16.5 g, 50 mmol) was dissolved in methanol, added with 0.4 g 10% Pd/C, and
hydrogenized at the normal temperature and pressure. When hydrogen was not
absorbed by the reaction mixture any more, Pd/C was filtered off. The filtrate was
concentrated to a small volume, and fed with dried HCl gas to generate a white solid,
which was collected by filtration and dried to give the title compound 8 (16.4 g. yield:
97%). The compound 8 (337 mg, 1 mmol) was stirred in the 0.4 N NaOH solution (5
mL, 2 mmol), and the precipitate was sollected by filtration, the cake was washed by
1
2 mLof water to give 9 as white solid (257 mg, yield: 86%). mp 79-80 °C. H NMR
(
CDCl 400 MHz): δ 11.27 (1H, br), 7.85 (1H, d, J = 2.8 Hz), 6.85 (2H, m), 4.08 (2H,
3,
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d, J = 6.4 Hz), 3.62 (2H, br), 2.68 (2H, q, J = 7.2 Hz), 2.59 (2H, q, J = 7.2 Hz), 1.96
1
3
(
2H, m), 1.31 (3H, t, J = 6.0 Hz), 1.15 (6H, m); C NMR (CD OD, 125 MHz): δ
3
1
65.64, 163.03, 153.04, 150.12, 141.48, 123.15, 120.24, 120.13, 116.62, 114.26,
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
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4
4
4
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5
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5
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0
1
2
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0
1
2
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0
1
2
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0
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0
1
2
3
4
5
6
7
8
9
0
+
71.11, 27.13, 22.24, 18.03, 12.38, 12.22, 9.60; LRMS m/z 302.3 [M+H] ; HRMS
+
(
ESI) m/z calculated for C H N O [M+H] 302.1897, found 302.1888; HPLC
1
7
23
3
2
purity 98.53%, t = 13.99 min.
r
N-(3-(5,6-Diethyl-4(3H)-pyrimidone-2-yl)-4-propoxyphenyl)propionamide (10).
Propanoic acid (58 mg, 0.78 mmol) was dissolved in dichloromethane (20 mL), added
with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI, 130 mg,
0.78 mmol), triethylamine (158 mg, 1.56 mmol) and stirred for 0.5 h.
1
-hydroxy-benzo-triazole (HOBT, 100 mg, 0.78 mmol) was added and stirred for 12
h, followed by addition of 8 (262 mg, 0.78 mmol). After stirred at room temperature
for 2 h, the reaction mixture was washed with water (10 mL) and saturated saline. The
organic phase was dried with anhydrous Na SO and concentrated under reduced
2
4
pressure to give an oil, which was purified by fast chromatography (ethyl
acetate/petroleum ether 1:10) to give the title compound as off white solid (250 mg,
1
yield: 90%). mp 172-173 °C. H NMR (CDCl 500 MHz): δ 8.25 (1H, s), 8.01 (1H,
3
,
dd, J = 9.0, 2.5 Hz), 7.81 (1H, s), 6.95 (1H, d, J = 9.0 Hz), 4.12 (1H, t, J = 7.0 Hz),
.64 (2H, q, J = 8.0 Hz), 2.58 (2H, q, J = 8.0 Hz), 1.94 (2H, q, J = 7.0 Hz), 1.25 (6H,
2
1
3
m), 1.13 (6H, m); C NMR (CDCl , 125 MHz): δ 172.56, 164.34, 162.54, 153.69,
3
1
51.36, 132.43, 125.38, 124.09, 122.16, 119.23, 113.28, 71.35, 30.46, 27.58, 22.41,
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2
2
2
2
2
2
2
3
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3
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5
5
5
5
5
6
+
18.71, 13.43, 13.15, 10.59, 9.75; LRMS m/z 380.3 [M+Na] ; HRMS (ESI) m/z
+
calculated for C H N O [M+H] 358.2125, found 358.2136; HPLC purity 97.02%,
2
0
28
3
3
tr = 16.46 min.
0
1
2
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7
8
9
0
1
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0
(2s)-1-Acetyl-N-(3-(5,6-diethyl-4(3H)-pyrimidone-2-yl)-4-n-propoxyphenyl)-2-py
rrolidine carboxamide (11). The title compound was prepared in a similar manner as
compound 10, using N-acetyl-L-proline instead of propanoic acid. Off-white solid
1
(
yield 66%). mp 141-143 °C. H NMR (CDCl 500 MHz): δ 9.63 (1H, s), 8.32 (1H, d,
3
,
J = 2.0 Hz), 7.84 (1H, dd, J = 9.0, 2.0 Hz), 6.92 (1H, d, J = 9.0 Hz), 4.78 (1H, d, J =
7.5 Hz), 4.10 (2H, t, J = 6.5 Hz), 3.63 (1H, m), 3.49 (1H, m), 2.70 (2H, q, J = 7.5 Hz),
2
.59 (3H, m), 2.17 (4H, m), 2.06 (1H, m),1.94 (3H, m), 1.30 (3H, t, J = 7.5 Hz), 1.15
1
3
(3H, t, J = 7.0 Hz), 1.11 (3H, t, J = 7.5 Hz); C NMR (CDCl , 125 MHz): δ 171.61,
3
1
1
69.15, 164.35, 162.51, 153.58, 151.54, 132.44, 124.84, 124.11, 122.12, 119.39,
13.16, 71.31, 60.40, 48.55, 27.53, 27.12, 25.12, 22.58, 22.44, 18.71, 13.41, 13.31,
+
10.62; LRMS m/z 441.4 [M+H] ; HRMS (ESI) m/z calculated for C H N O
2
4
33
4
4
+
[
M+H] 441.2496, found 441.2506; HPLC purity 98.51%, t = 14.37 min.
r
1-Methyl-N-(3-(5,6-diethyl-4(3H)-pyrimidone-2-yl)-4-n-propoxyphenyl)-4-piperi
dine carboxamide (12). The title compound was prepared in a similar manner as
compound 10, using 1-methyl-4-piperidinecarboxylic acid instead of propanoic acid.
1
Off-white solid (210 mg, yield 63%). mp 94-95 °C. H NMR (CD OD 500 MHz): δ
3
,
8
.17 (1H, d, J = 2.5 Hz), 7.84 (1H, dd, J = 9.0, 2.5 Hz), 7.26 (1H, d, J = 9.0 Hz), 4.15
(2H, 7, J = 6.5 Hz), 4.10 (2H, t, J = 6.5 Hz), 3.63 (2H, m), 3.15 (2H, m), 2.92 (3H, s),
.81 (3H, m), 2.65 (2H, q, J = 7.5 Hz), 2.21 (2H, m), 2.08 (2H, m), 1.87 (2H, m), 1.35
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1
3
(3H, t, J = 7.5 Hz), 1.20 (3H, t, J = 7.0 Hz), 1.05 (3H, t, J = 7.5 Hz); C NMR
(
CD OD, 125 MHz): δ 172.58, 162.94, 155.17, 153.78, 131.86, 126.53, 124.59,
3
1
22.37, 122.29, 116.84, 113.29, 70.81, 53.40, 51.10, 42.66, 39.93, 26.31, 24.69,
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
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5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
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2
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9
0
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6
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9
0
+
24.16, 22.07, 18.05, 12.20, 11.96, 9.41; LRMS m/z 427.4 [M+H] ; HRMS (ESI) m/z
+
calculated for C H N O [M+H] 427.2704, found 427.2697; HPLC purity 98.62%,
2
4
35
4
3
tr = 15.15 min.
-Acetylamino-N-(3-(5,6-diethyl-4(3H)-pyrimidone-2-yl)-4-n-propoxyphenyl)
4
butanamide (13). The title compound was prepared in a similar manner as compound
10, using 4-(acetylamino)-butanoic acid instead of propanoic acid. Off-white solid
1
(
228 mg, yield 68%). mp 142-143 °C. H NMR (CDCl 500 MHz): δ 9.01 (1H, s),
3
,
8.48 (1H, d, J = 2.5 Hz), 7.90 (1H, dd, J = 9.0, 3.0 Hz), 6.95 (1H, d, J = 9.0 Hz), 6.32
1H, s), 4.12 (2H, t, J = 6.5 Hz), 3.37 (2H, q, J = 4.8 Hz), 2.66 (2H, q, J = 7.5 Hz),
.57 (2H, q, J = 7.5 Hz), 2.43 (2H, t, J = 6.5 Hz), 2.01 (3H, s), 1.94 (4H, m), 1.29
(
2
1
3
(3H, t, J = 7.5 Hz), 1.13 (6H, m); C NMR (CDCl , 125 MHz): δ 171.68, 171.44,
3
1
63.98, 162.93, 153.66, 151.61, 132.63, 125.13, 123.96, 122.34, 119.30, 113.22,
71.36, 38.91, 34.70, 27.46, 26.16, 23.31, 22.44, 18.69, 13.41, 13.08, 10.63; LRMS
+
+
m/z 429.4 [M+H] ; HRMS (ESI) m/z calculated for C H N O [M+H] 429.2496,
2
3
33
4
4
found 429.2506.
2-Chloro-N-3-(5,6-diethyl-4(3H)-pyrimidone-2-yl)-4-n-propoxyphenyl)
acetamide (14). The compound 8 (0.74 g, 2 mmol) was dissolved in dichloromethane
(20 mL), added with triethylamine (2 mL), followed by slow addition of chloroacetyl
chloride (0.23 g, 2.05 mmol) under at 0°C. After stirred for 0.5 h, the reaction mixture
2
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was washed with water (10 mL), 1N HCl (5 mL), saturated sodium bicarbonate
solution (10 mL) and saturated saline respectively. The organic phase was dried
with anhydrous Na SO and concentrated. The resultant oil was recrystallized from
2
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0
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ethyl acetate-petroleum ether to give the compound 14 (0.71g, yield: 94%). mp
1
1
61-162 °C. H NMR (CDCl 500 MHz): δ 8.41 (1H, br), 8.35 (1H, d, J = 3.0 Hz),
3
,
7.95 (1H, dd, J = 9.0, 3.0 Hz), 7.03 (1H, d, J = 9.0 Hz), 4.22 (2H, s), 4.16 (2H, t, J =
6
.5 Hz), 2.69 (2H, q, J = 7.5 Hz), 2.58 (2H, q, J = 7.5 Hz), 1.97 (2H, m), 1.31 (3H, t, J
13
=
7.5 Hz), 1.13 (6H, m); C NMR (CDCl , 125 MHz): δ 164.09, 162.33, 154.558,
3
151.15, 151.14, 130.76, 125.56, 125.52, 124.52, 122.76, 119.49, 113.52, 71.49, 42.86,
7.53, 22.42, 18.74, 18.43, 13.39, 13.29, 10.63; LRMS m/z 378.3 [M+H]+.
2
N-(3-(5,6-Diethyl-4(3H)-pyrimidone-2-yl)-4-n-propoxyphenyl)-2-(4-methylpipera
zin-1-yl)acetamide (15). The compound 14 (0.38 g, 1 mmol) was dissolved in the
mixture of dimethylformide (3 mL) and triethylamine (202 mg, 2 mmol), and
N-methylpiperazine (100 mg, 1 mmol) was added. The reaction mixture was heated to
6
0 °C for 10 h, concentrated to dryness, washed with water and dried. The resultant
solid was recrystalllized from ethyl acetate/petrulem ether to obtain compound 15 as
1
off-white solid (278 mg, yield 63%). mp 134-136 °C. H NMR (CDCl 500 MHz): δ
3
,
1
1.17 (1H, s), 9.12 (1H, s), 8.28 (1H, d, J = 2.0 Hz), 7.98 (1H, d, J = 9.0 Hz), 7.02
(1H, d, J = 9.0 Hz), 4.15 (2H, t, J = 7.0 Hz), 3.18 (2H, s), 2.71 (6H, m), 2.60 (6H, m),
1
3
2
.38 (3H, s), 1.98 (2H, m), 1.32 (3H, t, J = 7.5 Hz), 1.14 (6H, m); C NMR (CDCl ,
3
125 MHz): δ 168.39, 164.34, 162.25, 153.92, 151.26, 131.64, 124.91, 124.41, 121.85,
19.66, 113.53, 71.43, 61.75, 55.07, 54.98, 53.21, 45.82, 27.65, 22.44, 18.73, 13.40,
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+
13.14, 10.64; LRMS m/z 442.4 [M+H] ; HRMS (ESI) m/z calculated for C H N O
2
4
36
5
3
+
[
M+H] 442.2813, found 442.2825; HPLC purity 98.80%, t = 11.98 min.
r
N-(3-(5,6-Diethyl-4(3H)-pyrimidone-2-yl)-4-n-propoxyphenyl)-2-(piperidin-1-yl)
acetamide (16). The title compound was prepared in a similar manner as compound
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
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0
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9
0
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2
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8
9
0
1
2
3
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9
0
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9
0
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2
3
4
5
6
7
8
9
0
1
5, using piperidine instead of N-methylpiperazine as off-white solid (360 mg, yield:
1
82.5%). mp 140-141 °C. H NMR (CDCl 500 MHz): δ 11.17 (1H, s), 9.28 (1H, s),
3
,
8
.29 (1H, d, J = 3.0 Hz), 7.99 (1H, dd, J = 9.0, 3.0 Hz), 7.02 (1H, d, J = 9.0 Hz), 4.16
(
2H, t, J = 6.5 Hz), 3.10 (2H, s), 2.69 (2H, q, J = 7.5 Hz), 2.60 (6H, m), 1.98 (2H, m),
1
3
1.67 (2H, m), 1.50 (2H, m), 1.32 (3H, t, J = 7.5 Hz), 1.14 (6H, m); C NMR (CDCl ,
3
1
25 MHz): δ 169.07, 164.47, 162.27, 153.83, 151.29, 131.84, 124.83, 124.36, 121.78,
119.66, 113.51, 71.41, 62.74, 54.99, 27.67, 26.23, 23.65, 22.44, 18.72, 13.41, 13.17,
+
1
0.64; LRMS m/z 427.4 [M+H] ; HRMS (ESI) m/z calculated for C H N O
2
4
35
4
3
+
[
M+H] 427.2704, found 427.2710; HPLC purity 98.50%, t = 19.02 min.
r
1-(3-(5,6-Diethyl-4(3H)-pyrimidone-2-yl)-4-n-propoxyphenyl)urea
(17).
The
compound 8 (200 mg, 0.6 mmol) was suspended in a mixed solution of water (5 mL)
and acetic acid (5 mL), added with potassium cyanate (81 mg, 1 mmol), and heated to
reflux and stirred for 2 h. The cooled reaction mixture was poured into water to
generate a white solid, which was washed with water (10 mL×3) and dried to give the
1
title compound 17 (210 mg, yield 91%). mp 144-146 °C. H NMR (DMSO-d , 500
6
MHz): δ 11.75 (1H, s), 8.58 (1H, s), 7.77 (1H, d, J = 2.5 Hz), 7.80 (1H, dd, J = 9.0,
2.5 Hz), 7.07 (1H, d, J = 9.0 Hz), 5.78 (2H, s), 4.01 (2H, t, J = 6.5 Hz), 2.58 (2H, q, J
=
7.5 Hz), 2.47 (2H, q, J = 7.5 Hz), 1.74 (2H, m), 1.20 (3H, t, J = 7.5 Hz), 1.05 (3H, t,
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